TOPOLOGY OF THE REGION SURROUNDING GLU(681) OF HUMAN AE1 PROTEIN, THEERYTHROCYTE ANION-EXCHANGER

AE1 protein transports Cl- and HCO3- across the erythrocyte membrane b y an electroneutral exchange mechanism, Glu(681) Of human AE1 may form part of the anion translocation apparatus and the permeability barrie r. We have therefore studied the structure of the sequence surrounding Glu(681), using scanning cysteine mutagenesis, Residues of the Ser(64 3) (adjacent to the glycosylation site) to Ser(690) region of cysteine less mutant (AE1C(-)) were replaced individually with cysteine, The ab ility of mutants to mediate Cl-/HCO3- exchange in transfected HEK293 c ells revealed that extracellular mutants, W648C, I650C, P652C, L655C, and F659C have an important role in transport. By contrast, only trans membrane mutation E681C fully blocked anion exchange activity, The top ology of the region was investigated by comparing cysteine labeling wi th the membrane-permeant cysteine-directed reagent 3-(N-maleimidylprop ionyl)biocytin, with or without prior labeling with membrane-impermean t lucifer yellow iodoacetamide (LYIA), Two regions readily label with 3-(N-maleimidylpropionyl)biocytin (Ser(643)-Met(663) and Ile(684)-Ser( 690)), We propose that poorly labeled Met(664)-Gln(683) corresponds to transmembrane segment 8 of AE1, Regions Ser(643)-Met(663) and Ile(684 )-Ser(690) localize, respectively, to extracellular and intracellular sites on the basis of accessibility to LYIA. On the basis of LYIA acce ssibility, we propose that the Arg(656)-Met(663) region forms a ''vest ibule'' that leads anions to the transport channel, Glu(681) is locate d 3 amino acids from the C terminus of transmembrane segment 8, which places the membrane permeability barrier within 5 Angstrom of the intr acellular surface of the membrane.